Diabetes mellitus and its associated complications are a major health problem in the developed world. Diabetics are 2- to 4-times more likely to have cardiovascular diseases (CVD) than general population. One feature of diabetes that has become apparent in recent years is excess oxidant stress. In preliminary data presented here, we have found that hyperglycemia and free fatty acids (FFA), two hallmarks of type I and type II diabetes, impart an oxidant stress in endothelial cells. These results in lipid peroxidation, tyrosine nitration of prostacyclin synthase (PGIS), reduced NO bioactivity, endothelial nitric oxide synthase (eNOS) uncoupling, and insulin resistance. We have also found that treatment with the AMP-activated kinase (AMPK) activator, 5-amino-4-imidazole carboxamide riboside (AICAR), prevents all of these events including the increase in oxidant stress and insulin resistance from occurring. A basic premise of this proposal is that AMPK activation could protect the endothelial cell against the adverse effects of hyperglycemia and FFA by increasing mitochondrial uncoupling protein (UCP)-2 that lead to a decrease in oxidant stress in parallel with an increase in NO bioactivity. Therefore, as a central hypothesis of this application, we propose that vascular diathesis of insulin resistance and diabetes is due, in part, from a hyperglycemia/FFA-induced oxidant stress and a compensatory activation of AMPK. The next part of our proposal will determine the consequences of AMPK activation on oxidant stress, endothelial function, and insulin signaling, capitalizing on preliminary data that AICAR reduces both cellular oxidant stress and insulin resistance from glucose and fatty acids in vitro and aortic lesions in Apo-E knockout (KO) enhanced by diabetes in vivo. In order to accomplish this goal, we propose to study 1). To determine if activation of AMPK by a number of means (pharmacological and molecular biological means) reduces oxidant stress and insulin resistance and to evaluate how it works, and 2). To determine if AMPK-dependent reduction in oxidant stress and endothelial dysfunction is operating in diabetes in vivo. This powerful combination of in vitro and in vivo techniques will provide novel information as to how the metabolic stresses associated with diabetes cause damage to the endothelium. They should also yield insights into how endothelium attempts to protect itself against these stresses and whether AMPK is a potential target for therapy for diabetes.